We have measured rates of uptake of arginine, glutamine, glutamate, serine, phenylalanine and glycine in Xenopus laevis oocytes cultured for periods of up to 24h in saline in the presence or absence of a mixture of 20 amino acids at concentrations approximating those in Xenopus plasma. Amino acid supplementation increased the total intracellular amino acid concentration from 8.2 to 18.4 nmol per oocyte. Specific Na(+)-dependent amino acid transporters (systems B0,+, Xag-) exhibit 'adaptive regulation' (up-regulation during amino acid deprivation and down-regulation during amino acid supplementation). Na(+)-independent transporters of glutamate, glutamine and glycine (including system asc) display an opposite modulation in activity, which may help to combat amino-acid-induced oxidative stress by increasing the supply of glutathione precursors. Single amino acids at physiological plasma concentrations (0.47 mmol l-1 L-alanine, 0.08 mmol l-1 L-glutamate) mimicked at least some effects of the amino acid mixture. The mechanisms of transport modulation do not appear to include trans-amino acid or membrane potential effects and, in the case of Na(+)-independent transport, are independent of protein or mRNA synthesis. Furthermore, activation of protein kinase C by phorbol 12-myristate 13-acetate did not significantly affect endogenous glutamine and glutamate transport. The Xenopus oocyte appears to possess endogenous signalling mechanisms for selectively modulating the activity of amino acid transport proteins expressed in its surface membranes, a factor for consideration when using oocytes as an expression system for structure-function studies of cloned amino acid transporters.
Sugar transporters in prokaryotes and eukaryotes belong to a large family of membrane proteins containing 12 transmembrane alpha-helices. They are divided into two classes: one facilitative (uniporters) and the other concentrative (cotransporters or symporters). The concentrative transporters are energised by either H+ or Na+ gradients, which are generated and maintained by ion pumps. The facilitative and H(+)-driven sugar transporters belong to a gene family with a distinctive secondary structure profile. The Na(+)-driven transporters belong to a separate, small gene family with no homology at either the primary or secondary structural levels. It is likely that the Na(+)- and H(+)-driven sugar cotransporters share common transport mechanisms. To explore these mechanisms, we have expressed cloned eukaryote Na+/sugar cotransporters (SGLT) in Xenopus laevis oocytes and measured the kinetics of sugar transport using two-electrode voltage-clamp techniques. For SGLT1, we have developed a six-state ordered model that accounts for the experimental data. To test the model we have carried out the following experiments. (i) We measured pre-steady-state kinetics of SGLT1 using voltage-jump techniques. In the absence of sugar, SGLT1 exhibits transient carrier currents that reflect voltage-dependent conformational changes of the protein. Time constants for the carrier currents give estimates of rate constants for the conformational changes, and the charge movements, integrals of the transient currents, give estimates of the number and valence of SGLT1 proteins in the plasma membrane. Ultrastructural studies have confirmed these estimates of SGLT1 density. (ii) We have perturbed the kinetics of the cotransporter by site-directed mutagenesis of selected residues.(ABSTRACT TRUNCATED AT 250 WORDS)